Measurement

Part:BBa_K337049

Designed by: Adlung L,Al Sabah J,Bayer P,Berrens R,Cristiano E,Flocke L,Kleinsorg S,Kolodziejczyk A,Macias Torre A,Mathur A,Mayilo D,Neumann S,Niopek D,Pisa R,Schad J,Schuhmacher L,Uhlig T,Wu X,Grimm D,Eils R   Group: iGEM10_Heidelberg   (2010-10-22)
Revision as of 09:32, 27 October 2010 by Stefan N (Talk | contribs)

pSMB_miMeasure

pSMB_miMeasure: basic scheme


pSMB_miMeasure is the standard fluorescent-based microRNA binding site characterization vector. It is composed of a mirrored construct setup (fig 1). Two fluorescent proteins EGFP and EBFP2 are driven by a bidirectional CMV promoter to ensure the same expression strength at both sides. The two fluorescent proteins are destabilized by fusion to a MODC degradation domain which leads to a protein half life of two hours. The shortened half life of the destabilized fluorescent proteins enables for time-laps experiments in order to analyze binding site properties in a dynamic microRNA environment. Furthermore the plasmid contains a BBb_2 (RFC12, RFC41) standard site behind the EGFP to enable user friendly exchange of binding site of choice. BamHI and HindIII sites behind the EBFP2 enable for cloning in a reference binding site. The plasmid further contains an ampicillin resistance for selection in bacteria. As the complete Plasmid has been synthesized by Geneart, it is in the pMA backbone (BB-2, RFC12).





Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 2643
    Illegal SpeI site found at 2657
    Illegal PstI site found at 2680
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal prefix found in sequence at 2643
    Illegal NheI site found at 2665
    Illegal PstI site found at 2680
    Illegal NotI site found at 2673
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 2643
    Illegal BamHI site found at 257
    Illegal XhoI site found at 2913
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 2643
    Illegal SpeI site found at 2657
    Illegal PstI site found at 2680
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 2643
    Illegal SpeI site found at 2657
    Illegal PstI site found at 2680
  • 1000
    COMPATIBLE WITH RFC[1000]


[edit]
Categories
Parameters
None